review article...

Hindawi Publishing CorporationInternational Journal of HepatologyVolume 2012, Article ID 859076, 7 pagesdoi:10.1155/2012/859076

Review Article

Biomarkers for Hepatocellular Carcinoma

Tara Behne1 and M. Sitki Copur1, 2

1 College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, USA2 Saint Francis Cancer Treatment Center, Grand Island, NE 68803, USA

Correspondence should be addressed to M. Sitki Copur, [email protected]

Received 16 October 2011; Accepted 27 February 2012

Academic Editor: Neil Guha

Copyright © 2012 T. Behne and M. S. Copur. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The hepatocellular carcinoma (HCC) is one of the most common malignant tumors and carries a poor survival rate. Themanagement of patients at risk for developing HCC remains challenging. Increased understanding of cancer biology andtechnological advances have enabled identification of a multitude of pathological, genetic, and molecular events that drivehepatocarcinogenesis leading to discovery of numerous potential biomarkers in this disease. They are currently being aggressivelyevaluated to establish their value in early diagnosis, optimization of therapy, reducing the emergence of new tumors, andpreventing the recurrence after surgical resection or liver transplantation. These markers not only help in prediction of prognosis orrecurrence but may also assist in deciding appropriate modality of therapy and may represent novel potential targets for therapeuticinterventions. In this paper, a summary of most relevant available data from published papers reporting various tissue and serumbiomarkers involved in hepatocellular carcinoma was presented.

1. Introduction

As molecular indicators of biological status, biomarkers,detectable in blood, urine, or tissue, can be useful for the clin-ical management of various disease states. Threshold concen-trations can be utilized to identify the presence of variousdiseases. Concentration fluctuations have the potential toguide therapy in disease progression. Numerous biomarkershave been identified for various disease states. Research isongoing to fully understand and evaluate the clinical signifi-cance of utilizing biomarkers. Time and money can besaved by avoiding empiric or broad treatment approachesto diseases of particular organs or systems, and ideally, bio-markers could serve as a measurement tool to detect dis-ease presence and progression and to guide more targetedtherapy. Many disease states, especially various types of can-cer, can be better understood by the utilization of tumor bio-markers. Hepatocellular carcinoma (HCC) is one such can-cer that can benefit from tumor biomarkers’ diagnostic, ther-apeutic, and prognostic capabilities.

HCC is the fifth most common malignant tumor andthe third leading cause of cancer-related deaths. Worldwide,there are about 626,000 new HCC cases and nearly 600,000HCC-related deaths each year with an incidence equal to thedeath rate [1, 2]. Although the molecular mechanisms bywhich HCC develops remain largely unclear, a multitude ofpathological, genetic, and molecular events that drive hepa-tocellular carcinogenesis has been identified.

Current gold standard and most commonly used bio-markers for patients at risk for HCC, alpha-fetoprotein(AFP) along with ultrasound every 6 to 12 months, is farfrom perfect. Serum AFP levels of more than 400 ng/mLare considered diagnostic; however, such high values areobserved only in a small percentage of patients with HCC.Ultrasound surveillance even performed at every threemonthly intervals cannot improve detection of small HCCbecause of limitations in recall procedures [3, 4].

With advances in understanding of tumor biology, alongwith the development of cellular and molecular techniques,the role of biomarkers related to early detection, invasiveness,

2 International Journal of Hepatology

metastasis, and recurrence has attracted great deal of researchinterest resulting in discovery and utilization of severalnovel markers in this disease. In this paper we try to givean overview of available data on this burgeoning area ofresearch.

2. Biomarkers for Liver Cancer

2.1. Oncofetal and Glycoprotein Antigens

2.1.1. Alpha-Fetoprotein (AFP). The first serologic assay fordetection and clinical followup of patients with hepatocellu-lar carcinoma was alpha-fetoprotein (AFP) which has beenthe standard tumor biomarker for HCC for many years. It isa glycoprotein produced by the fetal liver and yolk sac duringpregnancy. Serum AFP levels are often elevated in HCC,but this is not always the case. AFP levels may be elevatedinitially in the early stages of HCC and then drop or evennormalize before rising again as disease progression occurs[5]. Additionally, AFP elevation has also been recognizedin the presence of acute and chronic viral hepatitis as wellas in patients with cirrhosis caused by hepatitis C. Giventhe multiple indications that present with elevated AFPlevels, it is necessary to evaluate the significance of serumconcentrations. In general, consistently elevated serum AFPlevels greater than 500 ng/mL are indicative of HCC. Lowerserum concentrations which are only transient in nature aremore often present in benign liver disease [6]. If a patient hasknown risk factors for HCC, such as the presence of cirrhosis,increasing levels of AFP have been shown to correlate withthe development of HCC [6]. Unfortunately, AFP serumconcentrations do not correlate well with the prognostic val-ues of HCC such as tumor size, stage, or disease progression,and ethnic variability may also exist. Furthermore, in somecases of HCC, AFP elevations are not apparent at all [7]. TotalAFP can be divided into three different glycoforms, AFP-L1,AFP-L2, and AFP-L3-based on their binding capability tolectin Lens culinaris agglutinin (LCA). High percentageof AFP-L3 has been shown to be associated with poor dif-ferentiation and biologically malignant characteristics, worseliver function, and larger tumor mass [8].

2.1.2. Glypican-3. Glypican-3 (GPC3), a membrane-an-chored heparin sulfate proteoglycan, has been demonstratedto interact with growth factors and modulate their activities.It binds to the cell membrane through the glycosylphos-phatidylinositol anchors. GPC3 mRNA was upregulated sig-nificantly in tumor tissues of HCC compared to paraneoplas-tic liver tissue, liver tissues of healthy adults, and liver tissuesof patients with nonmalignant hepatopathy. The expressionof GPC3 (at both mRNA and protein levels) in the serum ofHCC patients was significantly higher than that in the serumof healthy adults or patients with nonmalignant disease. Itcan be detected in 40–53% of HCC patients and 33% ofHCC patients seronegative for both AFP and Des-gamma-carboxyprothrombin (DCP) [9, 10]. It has been shownthat soluble GPC3 (sGPC3), the NH2-terminal portion ofGPC3, is superior to AFP in the sensitivity of detecting well

Table 1: Diagnostic values of HCC serum markers [12–14].

Type of testSensitivity

(%)Specificity

(%)

AFP-L3 61.6 92.0

DCP 72.7 90.0

AFP 67.7 71.0

AFP-L3 + DCP 84.8 97.8

AFP-L3 + AFP 73.7 86.6

DCP + AFP 84.8 90.2

AFP-L3 + DCP + AFP 85.9 59.0

or moderately differentiated HCC, and the simultaneousdetermination of both markers improves overall sensitivityfrom 50% to 72%. Recently, a study compared the survivalrate between the GPC3-positive and GPC3-negative HCCpatients. GPC3 positivity correlated with poor prognosis andidentified as an independent prognostic factor for the overallsurvival on multivariate analysis [11].

2.2. Enzymes and Isoenzymes

2.2.1. Des-Gamma-Carboxy (Abnormal) Prothrombin (DCP).DCP is produced by the malignant hepatocyte and appearsto result from an acquired posttranslational defect in thevitamin-K-dependent carboxylase system. DCP productionis independent of vitamin K deficiency, although phar-macological doses of vitamin K can transiently suppressDCP production in some tumors. DCP levels greater than0.1 AU/mL (100 ng/mL) on ELISA are highly suggestive ofHCC or tumor recurrence. Normalization of DCP levelscorrelates well with successful tumor resection and appearsto be an excellent marker of tumor activity. It is thought thatthe combination of AFP and DCP assays will increase thesensitivity of testing. The correlation between tumor size andDCP levels is not yet clearly defined. It appears that thereis a correlation in DCP levels and large tumors; however,the same is not the case in small tumors (<3 cm) [15].A cross-sectional case control study involving 207 patientsdetermined that DCP is more sensitive and specific than AFPfor differentiating HCC from nonmalignant liver disease. Inthis study there were 4 groups studied: normal healthy sub-jects; patients with noncirrhotic chronic hepatitis, patientswith compensated cirrhosis, and patients with histologicallyproven HCC. Both DCP and AFP levels increased among thegroups as disease severity increased (from normal to HCC),but DCP values had less overlap among the groups than AFP.Study results concluded that a DCP value of 125 mAU/mLyielded the best sensitivity and specificity for differentiatingpatients with HCC from those with cirrhosis and chronichepatitis [16]. Sensitivity and specifity of total AFP, AFPglycoforms, DCP, and combinations of both markers havebeen summarized in Table 1.

2.2.2. Gamma-Glutamyl Transferase. Serum gamma-gluta-myl transferase (GGT) in healthy adults is mainly secretedby hepatic Kupffer cell and endothelial cell of bile duct,

International Journal of Hepatology 3

and its activity increases in HCC tissues. Total GGT can bedivided into 13 isoenzymes by using polymer acrylamidegradient gel electrophoresis, and some of them can onlybe detected in the serum of HCC patients. Sensitivities ofGGTII have been reported to be 74.0% in detecting largeHCC and 43.8% in detecting small HCC. Sensitivity can besignificantly improved with the simultaneous determinationof GGTII, DCP, and AFP [17].

2.2.3. Serum Alpha-1-Fucosidase. Alpha-l-fucosidase (AFU)is a lysosomal enzyme found in all mammalian cells witha function to hydrolyze fucose glycosidic linkages of gly-coprotein and glycolipids. Its activity increases in the se-rum of HCC patients (1418.62 ± 575.76 nmol/mL/h) com-pared with that in the serum of healthy adults (504.18 ±121.88 nmol/mL/h, P < 0.05), patients with cirrhosis(831.25 ± 261.13 nmol/mL/h), and patients with chronichepatitis (717.71± 205.86 nmol/mL/h). It has been reportedthat the sensitivity and specificity of AFU at the cut-offvalue of 870 nmol/mL/h were 81.7% and 70.7%, respectively[18]. AFU measurement is useful in association with AFPin early diagnosis of HCC and could serve as a valuablesupplementary to AFP. It has been indicated that HCC willdevelop within few years in 82% of patients with liver cir-rhosis, if their serum AFU activity exceeds 700 nmol/mL/h.The activity of AFU was reported to be elevated in 85% ofpatients at least 6 months before the detection of HCC byultrasonography [19].

2.2.4. Human Carbonyl Reductase 2. This enzyme expressedin the human liver and kidney is important in detoxificationof the reactive alpha-dicarbonyl compounds and reactiveoxygen species deriving from oxidative stress in HCC. Thehuman carbonyl reductase 2 levels have been shown to beinversely correlated to the pathological grading of HCC [20].

2.2.5. Golgi Phosphoprotein 2. Golgi phosphoprotein 2(GOLPH2), a Golgi-apparatus-associated protein, has beenshown to have a higher sensitivity than AFP in the detectionof HCC [21]. A recent study found that GOLPH2 proteinwas highly expressed in tissues of HCC (71%) and bileduct carcinoma (85%) patients. GOLPH2 protein levels weredetectable and quantifiable in sera by ELISA. In patientswith hepatitis C, serial ELISA measurements in the course ofthe disease appear to be a promising complimentary serummarker in the surveillance of HCC [22].

2.3. Growth Factors and Their Receptors

2.3.1. Transforming Growth Factor-Beta (TGF-Beta). Belong-ing to a superfamily of polypeptide signaling molecules in-volved in regulating cell growth, differentiation, angiogen-esis, invasion, and immune function, TGF-beta is a predomi-nant form of growth factor family in humans. Its mRNAand protein are overexpressed in HCC compared with sur-rounding liver tissues, especially in small and well-differen-tiated HCCs [23]. However, no relationship has been shownbetween TGF-beta expression and posthepatectomy survival

[24]. Serum TGF-beta level has been found to be elevated inHCC patients compared to healthy adults or patients withnonmalignant liver disease [25–27].

2.3.2. Tumor-Specific Growth Factor (TSGF). Malignant tu-mors release tumor-specific growth factor (TSGF) intoperipheral blood during their growing period. Serum levelsof TSGF may reflect the existence of tumor. TSGF can be usedas a diagnostic marker in detecting HCC, and its sensitivitycan reach 82% at the cut-off value of 62 U/mL and may havea higher accuracy with the simultaneous determination ofother tumor markers. The simultaneous determination ofTSGF (at the cut-off value of 65 U/mL), AFP (at the cut-offvalue of 25 ng/mL), and serum ferritin (at the cut-off valueof 240 ng/mL) can reach a sensitivity and specificity of 98.4%and 99%, respectively [26].

2.3.3. Epidermal Growth Factor Receptor Family. The epider-mal growth factor receptor (EGFR) family consists of fourclosely related transmembrane tyrosine kinase receptors:EGFR (erbB-1), c-erb-2 (Her-2/neu), c-erb-3 (HER-3), andc-erb-4 (HER-4). These bind with ligands of the EGFfamily, including EGF, TGF-alpha, and heparin-binding EGF.High levels of EGFR expression have been associated withearly recurrence and reduced disease-free survival followingresection of hepatocellular carcinoma [27].

2.3.4. Hepatocyte Growth Factor/Scatter Factor. Hepatocytegrowth factor/scatter factor (HGF/SF) is a cytokine with awide range of effects from embryonic development and liverregeneration. It is associated with molecular mechanismsof hepatocarcinogenesis via paracrine system involving itscellular receptor, c-met. High c-met expression has beenshown in invasive-type HCC and has been associated withmetastasis and reduced overall survival [28, 29].

2.3.5. Basic Fibroblast Growth Factor. This is a soluble hepa-rin-binding polypeptide with a potent mitogenic effect onendothelial cells. Elevated levels above the median of>10.8 pg/mL have been shown to predict decreased disease-free survival [30]. Recent preliminary data with targetedtherapy lenalidomide which inhibits fibroblast growth factor(FGF) showed promising and in some patients dramaticactivity in HCC patients [31].

2.4. Molecular Markers

2.4.1. Circulating Nucleic Acids: mRNAs. The analysis of cir-culating nucleic acids in plasma offers another avenue fornoninvasive monitoring of a variety of physiological andpathologic conditions [30, 31]. Numerous applications basedon the detection of circulating cell-free nucleic acids inhuman plasma have been reported for the management ofmalignancies. The fundamental principle underlying theseapplications relates to the detection in plasma of extracel-lular nucleic acid molecules derived from diseased organs.Analysis of cell-free plasma RNA offers an opportunity forthe development of pathology-related markers [32–34].


Alpha-Fetoprotein mRNA (AFP mRNA). Matsumura et al.first reported that single HCC cell could be detected in cir-culation by means of reverse-transcription polymerase chainreaction (RT-PCR), targeting AFP mRNA [35]. This led tofurther reports of the value of AFP mRNA as a predictor forHCC recurrence. Rather controversial results were attributedto the blood borne dispersion of both tumor cells and normalliver cells and the mistranscription of mRNA encodingAFP by peripheral mononuclear cells. The recurrence-freeinterval of HCC patients with postoperative serum AFPmRNA positivity has been reported to be significantly shorterthan that of HCC patients with postoperative negativity(53% versus 88% at 1 year, 37% versus 60% at 2 years,P = 0.014) [34] and (52.6% versus 81.8% at 1 year, 15.6%versus 54.5% at 2 years, and 0% versus 29.2% at 3 years,P < 0.001) [36]. A meta-analysis showed that the expressionof AFP mRNA one week after surgery was correlated with therecurrence of HCC [37].

Gamma-Glutamyl Transferase mRNA (GGT mRNA). Similarto AFP, GGT mRNA can be detected in the serum and livertissues of healthy adults, patients with liver disease, benignliver tumor, HCC, and secondary tumors of the liver [38].The two types of GGT mRNA, type A and type B, have beenidentified. Type B is the predominant one in cancerous tissuesuggesting that changes in the expression of hepatic GGTmRNA may be related to the development of HCC [39].Patients with HCC harboring type B GGT mRNA both incancer and in noncancerous tissue had a worse outcome,earlier recurrence, and more recurrence-related mortality.The presence of type B GGT mRNA in cancerous tissue wasstatistically correlated with high serum level of AFP, daughternodules, higher postresection recurrence rate than thosewithout it (63.6% versus 14.3%), and lower postrecurrencesurvival. The presence of type B GGT mRNA in non-cancerous liver tissue was significantly correlated with hep-atitis C infection, high serum level of AFP, absence of infil-tration of capsule, vascular permeation, daughter nodules,postresection recurrence, and postrecurrence survival [40].

Insulin-Like Growth Factor II (IGF-II) mRNA. Abnormalexpression of IGF-II mRNA can be a useful tumor markerfor diagnosis, differentiation, extrahepatic metastasis, andmonitoring of postoperative recurrence in HCC. The deter-mination of serum insulin-like growth factor-II (IGF-II) (atthe cut-off value of 4.1 mg/g, prealbumin) has a sensitivityof 63%, specificity of 90%, and accuracy of 70% in thediagnosis of small HCC [41]. It can be a complementarytumor marker to AFP for diagnosis of small HCC. Thesimultaneous determination of IGF-II and AFP (at the cut-off value of 50 ng/mL) can improve the sensitivity to 80% andaccuracy to 88% [42].

Albumin mRNA. Albumin is the most abundant proteinin the body synthesized by the liver. mRNA of albumin isdetectable in human plasma and could be a diagnosticallysensitive marker for liver pathologies. Extracellular-basedassays (circulating DNA/RNA) have been found to be better

than cell-based assays (circulating tumor cells) in detectionof preneoplastic lesions and micrometastases as plasma levelsof circulating cancer-derived nucleic acid are higher thanthe levels of circulating cancer cells and are less proneto sampling errors. Cheung and colleagues studied thepreoperative plasma samples obtained from 72 HCC patientswho had undergone liver transplantation and found thatpatients with plasma albumin mRNA level (>14.6) had asignificantly higher recurrence rate on multivariate analysis.High plasma albumin mRNA level predicted the 2-yearrecurrence rate with sensitivity and specificity of 73% and70%, respectively [43].

MicroRNAs (miRNAs). MicroRNAs (miRNAs) are a familyof endogenous, small (21–23 nucleotides), noncoding butfunctional RNAs, which have been found in worms, flies,and mammals including human beings [44]. It is estimatedthat there are about 1,000 miRNA genes in the humangenome with approximately 500 miRNA genes being alreadyidentified [45]. Similar to mRNA, HCC-associated miRNAscould be used as diagnostic and prognostic biomarkers ofHCC with a potential for even greater accuracy. MiRNAscan accurately predict whether liver cancer will spread andwhether liver cancer patients will have shorter or longersurvival. MicroRNAs regulate gene expression by binding tospecific messenger RNAs and prevent their translation intoprotein. Because each type of miRNA is able to downregulatehundreds of genes at a time, they can control entire tran-scriptional programs that determine fundamental cellularproperties and behavior. Accordingly, miRNA profiling hasemerged as an extremely valuable method for phenotypingand subclassifying tumors [44]. Compared to conventionalgene expression profiling (in which protein-coding, mes-senger RNAs are examined), miRNA analysis has severaladvantages. Due to the stability of miRNAs, formalin-fixedsamples (rather than frozen tissue) can be used. Further-more, the interrogation of hundreds of miRNAs (and oftensignificantly fewer) yields as much information as might begleaned from examining thousands of messenger RNAs.

Many independent groups have conducted compre-hensive analyses of miRNAs in HCC, and a plethora ofinformation on miRNA markers has been identified. Many ofthese miRNA signatures correlate with important biologicalparameters, such as metastasis [46–48], differentiation [49–51], HBV or HCV infection [52, 53], tumor recurrence[54], and patient survival [55, 56]. Some miRNAs areinvolved in HCC carcinogenesis by promoting cancer stemcell and by controlling cell proliferation and apoptosis;others are associated with HCC progression by controllingcell migration and invasion. These HCC-associated miRNAsnot only provide new insights into the molecular basisof HCC but also serve as new tools for HCC diagnosisand prognosis. Currently a few miRNA signatures, however,could potentially be used in this area. Some miRNAs havebeen validated in an independent cohort, paving the way forclinically useful platforms to assess HCC risk and outcome.This promising area of research awaits further validation inprospective studies [57].


Table 2: Various HCC biomarkers and their clinical use.

HCC marker Clinical use

Alpha-fetoprotein Early diagnosis, monitoring, and recurrence

Lens culinaris agglutinin reactive AFP (AFP-L3%) Early diagnosis and prognosis, vascular invasion

Des-gamma-carboxy prothrombin (DCP) Early diagnosis and prognosis, portal vein invasion and metastasis

Gamma-glutamyl transferase Early diagnosis complementary to other markers

Alpha-l-fucosidase Early diagnosis

Glypican-3 Early diagnosis

Human carbonyl reductase 2 Prognosis

Golgi phosphoprotein 2 Tumor aggressiveness

Transforming growth factor beta Tumor invasiveness

Hepatocyte growth factor (HGF) Prognosis and disease recurrence

Transforming growth factor-b (TGF-b) Prognosis invasiveness

Tumor specific growth factor Diagnosis complementary to other markers

Epidermal growth factor receptor family Early recurrence

Hepatocyte growth factor Metastasis reduced survival

Micro RNAs Tumor spread and survival

2.5. Pathological Biomarkers. Finally there have been reportsof pathological biomarkers of HCC for diagnosis and pro-gnosis. Some of these diagnostic biomarkers focus on immu-nochemical staining patterns to distinguish high-grade dys-plastic nodules and well-differentiated HCC. The best typeof immunostaining for this difficult condition has beenreported to be the combination of heat-shock protein 70(HSP70), glypican-3 (GPC3), and glutamine synthetase(GS). For prognostic use a number of histological and immu-no-histochemical markers such as markers of cell prolifera-tion (Ki67), apoptosis or cell survival (survivin), cell adhe-sion molecules (E-cadherin), neoangiogeneis (VEGF), andmore have been looked in small studies showing promise;however, most of these markers have not been validated inlarge studies [57]. Various HCC biomarkers and their clinicaluse have been summarized in Table 2.

3. Discussion

Hepatocarcinogenesis is a complex multistate process usuallyoccurring after many years of chronic exposure to sev-eral mitogenic and mutagenic environments precipitatingrandom genetic alterations. Recent evidence suggest thatintrinsic biologic characteristics of the tumor in termsof proliferation and invasiveness are probably related todifferent composition and activity of the microenvironment,leading to very different clinical outcomes. HCC is ratherunique with its ability to synthesize various tumor-relatedproteins rendering itself more suitable to biomarker-relatedresearch than other tumors. Because of the large multitude ofbiomarkers reported in this disease, selecting the biomarkerswhich would be most useful in clinical practice has beenmore than challenging. In this rather brief overview, we triedto focus on most widely used and accepted biomarkers.

Despite its limitations, serum AFP still remains themost widely used tumor marker in clinical practice. Recent

research favors the circulating hepatoma-specific AFP sub-fraction AFP-L3 and DCP over AFP alone in differentiatingHCC from nonmalignant hepatopathy and detecting smallHCC. Furthermore, some other tumor markers, such asGPC3, GGT II, AFU, have been shown to be supplementaryto AFP and DCP in the detection of HCC. Some of themeven can be detected in HCC patients seronegative forboth AFP and DCP, thus indicating that the simultaneousdetermination of these markers may improve the accuracy.

However, most exciting and promising area of researchin this disease has been the identification of a new group ofmolecules called miRNAs. MiRNAs have been discovered tobe aberrantly expressed in HCC, and some of them are func-tionally involved in HCC carcinogenesis and progression.Furthermore, certain microRNAs are associated with HCCor related to HCC subtypes, implicating the potential use ofmicroRNAs in HCC patient stratification of diagnosis andprognosis. Some of these HCC-associated miRNAs have beenvalidated in independent cohorts. This brings the possibilityof developing clinically useful platforms to develop HCCdiagnosis, risk assessment, and patient risk stratification withthe ultimate goal of personalized therapy.

4. Conclusion

Research into the molecular biology of hepatocarcinogenesishas identified numerous biomarkers which could provideadditional information for HCC biologic behavior metastasisand recurrence to that gained from traditional histopatho-logical features. A large number of biomarkers have beenshown to have potential predictive significance. However,most of them have been studied retrospectively. Effortsshould be directed towards prospective clinical trials in eval-uating the prognostic significance of these markers. Thesemolecules not only help in prediction of prognosis for pa-tients with HCC but may also assist in deciding appropriate


modality of therapy and represent novel targets for therapeu-tic interventions.

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